1. Field of the Invention
The present invention relates to methods for improved baculovirus insecticides by expanding the host range against insect pests.
2. Description of Related Art
Baculoviruses are insect-specific viruses that can be used as microbial insecticides. They are considered an attractive alternative to chemical insecticides because in contrast to many chemicals they do not leave potentially harmful chemical residues in the environment nor do they harm vertebrate animals or beneficial insects. Baculovirus insecticides comprised of nuclear polyhedrosis viruses (NPVs), a subgroup of baculoviruses, were first registered by the U.S. Environmental Protection Agency in 1979. However several features of natural baculovirus isolates detract from their positive attributes as insecticides. One of these is a prolonged period between ingestion of the virus, the natural route of infection, and the cessation of insect feeding. Another is that baculoviruses have restricted host ranges, infectivity is usually limited to members of the same genus or family of the original host (Groner, A., Specificity and safety of baculoviruses., In Granados, R. R. and Federici, B. A. (eds), CRC Press, Boca Raton, Fla., The biology of baculoviruses., Vol. I, pp 177-202 (1986)). The mechanisms that determine baculovirus host specificity are unknown. Because of this several different baculovirus insecticides may be required to control insect pests if several species are infesting a crop. Baculoviruses, in particular Autographa californica nuclear polyhedrosis virus (AcMNPV), have been studied intensively at the molecular level. As a result of these studies, methods have been developed which permit modification of these viruses by genetic engineering. Recently, a great deal of effort has been focused on the improvement of baculovirus insecticides. These efforts have focused primarily on reducing the time to stop insect feeding following ingestion of a virus. This has been accomplished by the insertion of genes for insecticidal proteins (Bonning, B. C., et al, Insect Blochem. Molec. Biol. 22:453-458 (1992); Carbonell, et al, Gene 73:409-418 (1988); Eldridge, R., et al, Appl. Environ. Microbiol. 58:1583-1591 (1992); Hammock, B. D., et al, Nature (London), 344:458-461 (1990); Maeda, S., Biochem. Biophys. Res. Commun. 165:1177-1183 (1989); Maeda et al, Virol. 184:777-780 (1991); Martens, J. W. M., et al, Appl. Environ. Microbiol. 56:2764-2770 (1990); McCutchen, B. F., et al., Bio/Technology 9:848-852 (1991); Merryweather, A. T., et al, J. Gen. Virol. 71:1535-1544 (1990); Stewart, L. M., et al, Nature (London), 352:85-88 (1991); Tomalski, M. D. and Miller, L. K., Nature (London), 352:82-85 (1991); Tomalski, M. D. and Miller, L. K., Biotechnology 10:545-549 (1992)) or by the deletion of a baculovirus gene, ecdysteroid UDP-glucosyl transferase (EGT), that prolongs insect feeding (O'Reilly, D. R. and Miller, L. K., Science 245:1110-1112 (1989); O'Reilly, D. R., et al., Bio/Technology 9:1086-1089 (1991)). In order to modify the host range of a baculovirus it is necessary to identify the genes that control baculovirus host range so that the viruses can be altered to infect a variety of insect pest species.
The NPVs that infect lepidopteran larvae, the caterpillar stages of moths and butterflies, produce two morphological forms of the virus. A budded form that spreads the virus to tissues within the insect is used for propagating virus in insect cells. An occluded form in which the virions are packaged within a protein crystal called polyhedra is the normal infection route for larvae. The polyhedra are composed primarily of a single polypeptide called polyhedrin. The virus is acquired when the caterpillars eat foliage that is contaminated with polyhedra. The polyhedra dissolve in the midgut of the insect and release the virions which then enter the midgut cells to begin the infection. In baculovirus expression systems that are used to produce large quantities of various proteins for research or to produce commercial products such as pharmaceuticals the polyhedron gene, which is not necessary for virus growth in cell culture, is replaced by the gene of the protein to be produced and no polyhedra are produced. However, baculovirus expression systems have also been developed that permit expression of a foreign gene while maintaining the polyhedrin gene and thus the ability to produce occluded viruses (polyhedra) for insecticidal use.
The prior art has described various recombinant baculovirus. U.S. Pat. Nos. 5,180,581 to Miller et al; 5,266,314 to Maeda and 5,352,451 to Miller et al describe various recombinant baculovirus including foreign DNA which provide insect control. U.S. Pat. No. 5,004,687 to Miller describes a vital expression vector for production of exogenous gene products in insects. Various promoters are used, such as Rous sarcoma virus long terminal repeat promoter (LTR-RSV) genes can include toxic genes. Endogenous promoters include early and delayed early promoters of AcMNPV. Also the polyhedron promoter was described. U.S. Pat. No. 4,745,051 to Smith and Summers describes a method for producing a recombinant baculovirus expression vector which expresses a gene in a host. This general method is used in the present invention and is not repeated in the present specification.